The present invention is directed in general toward bioassays, biosensors, and cell-based assays, and more specifically to systems and system components that include living, engineered immunocytes that express fluorescent fusion reporter proteins for use in multiplexed bioassays for the detection of target analytes.
Biosensor systems such as those disclosed in U.S. Pat. Nos. 9,023,640; 9,752,199; 9,850,546; 9,850,547; and 9,850,548; and U.S. patent application Ser. Nos. 15/848,104; and 62/572,617, all of which are incorporated by reference herein, in their entirety, for all purposes, may include the following elements or components: (i) a living biological cell of a predetermined type (e.g., an engineered immunocyte); (ii) a signal-generating reporter within the living biological cell, wherein the signal-generating reporter is responsive to predetermined changes occurring within the living biological cell; (iii) a signal transduction pathway or activator mechanism associated with the signal-generating reporter, wherein the signal transduction pathway or activator mechanism is operative to induce the predetermined changes within the living biological cell; (iv) a universal detector element (e.g., a transmembrane, non-antibody fusion protein expressed by the living immunocyte) associated with the activator mechanism, wherein the universal detector element is operative to trigger the activator mechanism; (v) an analyte binding element (e.g., a soluble antibody) associated with the universal detector element, wherein the analyte binding element is specific to both the universal detector element and a target analyte; and (vi) wherein upon the binding of an analyte binding element to which a target analyte is also bound to the universal detector element, the universal detector element triggers the activator mechanism causing the predetermined changes to occur within the living biological cell, thereby causing the signal-generating reporter to generate a detectable signal. In such systems, the detectable signal may be a flash of visible light. Additionally, transmembrane, non-antibody fusion proteins expressed by the living immunocyte in such systems may include (a) at least one component of a protein that is adapted to bind to the analyte binding element (e.g., a bacterial binding protein or antibody binding domain derived from an Fc receptor protein); and (b) at least one component of a receptor complex normally expressed on the surface of the immunocyte (e.g., IgM; Igα/β; IgE; CD19; or CD3).
Other detection systems such as those described in U.S. Patent Application Nos. 62/643,378; and 62/651,916, which are incorporated by reference herein, in their entirety, for all purposes, may include the following elements or components: (i) a programmable receptor complex expressed by a living, engineered immunocyte (e.g., CD4+ T cell, CD8+ T cell, γδ T cell, or allogeneic cell); (ii) wherein the programmable receptor complex includes a plurality of native or endogenously-expressed receptor subunits (e.g., T cell receptor subunits such as CD3-epsilon, CD3-delta, CD3-gamma, TCR alpha, TCR beta, and CD3-zeta); (iii) wherein at least one of the plurality of native or endogenously-expressed receptor subunits has been engineered or modified to include either a biotin-binding component (e.g., monomeric streptavidin 2 or enhanced monoavidin) or an FcγRI receptor component; and (v) wherein the biotin-binding component or FcγRI receptor component is operative to bind to a target detector molecule that binds to or otherwise interacts with a predetermined target. Such detection systems have diagnostic applications and may also include within the living, engineered immunocyte a signal-generating reporter that emits a detectable signal upon binding to a target analyte.
While the systems described above are highly effective for their intended purposes, the signal-generating reporter included therein is capable of reporting only a single type of binding event. This aspect of the systems described above limits the use of these detection systems and makes them unsuitable for multiplexing applications that are directed toward the detection of multiple targets or multiple types of binding events. Accordingly, there is a need for a single biosensor system or single living-cell based detection system that can simultaneously detect the binding of different types of targets, or that can simultaneously detect different types of binding events, under the same test conditions.